Carboxy-terminal-extended variant of the human fibrinogen alpha subunit: a novel exon conferring marked homology to beta and gamma subunits.

Similarities between the N-terminal regions of the three subunits of the clotting protein fibrinogen--(alpha beta gamma)2--suggest that they evolved from a common progenitor. However, to date no human alpha chain has been found with the strong C-terminal homology shared by the beta and gamma chains. Here we examine the natural product of a novel fibrinogen alpha chain transcript bearing a separate open reading frame that supplies the missing C-terminal homology to the other chains. Additional splicing leads to the use of this extra sequence as a sixth exon elongating the alpha chain by 35%. Since the extended alpha chain (alpha E) is assembled into fibrinogen molecules and its synthesis is enhanced by interleukin-6, it suggests participation in both the acute phase response and normal physiology.

Noncoordinate synthesis of the fibrinogen subunits in hepatocytes cultured under hormone-deficient conditions.

Differential detergent gel electrophoresis conditions are described which enable the accurate quantitation of radiolabel incorporated into each of the closely migrating, constituent polypeptides of chicken fibrinogen: glycosylated and nonglycosylated A alpha, B beta, gamma', and gamma. These methods were applied to analysis of fibrinogen synthesis by monolayer cultures of chick embryo hepatocytes to determine whether the cells coordinate biosynthesis of the fibrinogen subunits under nonstimulated or basal conditions (i.e. in the absence of hormones) and in the presence of serum, which is a potent stimulator of fibrinogen production. Since secretion of the subunits apparently depends on their oligomeric assembly into the general structure (A alpha, B beta, gamma)2, it was thought that their synthesis might be stoichiometric. Incorporation of [35S]methionine into the subunit chains was determined for both cellular and secreted fibrinogen, immunoprecipitated from pulse-labeled and continuously labeled cultures. Molar ratios of subunit synthesis and the degree of serum-induced stimulation for each subunit were calculated. Specific subunit mRNA levels were also evaluated with a cell-free translation assay as well as microinjection of RNA into Xenopus oocytes. The results indicate, to the contrary, that in hormone-deprived hepatocytes there is a deficiency in A alpha chain synthesis, correlating with reduced A alpha-specific mRNA levels, which leads to hepatocellular degradation of surplus B beta and gamma chains. Addition of serum to the cellular environment, while increasing rates of subunit synthesis, also corrects the deficiency in A alpha chain synthesis, thereby restoring a measure of balance and preventing much of the degradation. The outcome of this serum-induced enhancement and coordination of fibrinogen subunit gene expression is a dramatic (more than 20-fold) stimulation of fibrinogen secretion.

Selective intracellular degradation of fibrinogen and its reversal in cultured hepatocytes.

Primary monolayer cultures of chick embryo hepatocytes were employed in pulse-chase experiments to examine plasma protein synthesis and secretion. The fates of [35S]methionine-labeled fibrinogen and transferrin were monitored in cell extracts and in spent culture media. It was found that hepatocytes, which were maintained in the absence of added hormones or serum, released into the medium virtually all of the label of transferrin but only 30% of the label in fibrinogen. The remainder of the labeled fibrinogen was retained by the cells, gradually disappearing in a manner suggestive of its intracellular degradation. To stimulate fibrinogen production on as many levels as possible, fetal bovine serum was added to the medium of the cultured cells. Serum elicited an increase in the level of fibrinogen mRNA which was accompanied by a 7-fold increase in the rate of fibrinogen synthesis as well as the complete release of fibrinogen label, resulting in an overall 20-fold enhancement in the hepatocellular output of this protein. Thus, both the amount of fibrinogen synthesized as well as the amount ultimately secreted are subject to modulation by the hepatocellular environment.

Glycosylated A alpha chains in chicken fibrinogen.

Fibrinogen immunoprecipitated from cultured chick embryo hepatocytes was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and compared with fibrinogen from chicken plasma. The character and relatedness of the constituent polypeptide bands were established on the basis of enzymatic treatment, peptide analysis, metabolic labeling with [14C]glucosamine, and inhibition of glycosylation with tunicamycin. Hepatocyte-derived fibrinogen resolved into five polypeptides that, in order of decreasing apparent molecular weight, were identified as glycosylated A alpha, non-glycosylated A alpha, B beta, gamma', and gamma. Fibrinogen immunoprecipitated directly from chicken plasma yielded an identical profile except for an additional smaller A alpha chain. This small A alpha chain appears to be the product of partial proteolysis in the circulation and was the only A alpha band found in purified plasma fibrinogen (fraction I-2). The observation of glycosylated A alpha chains is novel. The gamma/gamma' chain heterogeneity appears to be due to an amino acid extension similar to that observed in mammalian fibrinogens. Fibrinogen from cells exposed to fetal bovine serum, a potent stimulator of fibrinogen production, was enriched in glycosylated A alpha chains, which constituted approximately one-third of the A alpha chain population. Serum did not affect the gamma/gamma' chain distribution.

Selective block of albumin gene expression in chick embryo hepatocytes cultured without hormones and its partial reversal by insulin.

Rates of synthesis of albumin and transferrin were compared with the levels of their cognate mRNAs in primary monolayer cultures of chick embryo hepatocytes over a period of 72 h. These liver cells were exposed, from the onset of culture, only to a chemically defined medium devoid of hormonal or macromolecular supplement. Synthesis of transferrin was constant whereas synthesis of albumin diminished to near 0 in 3 days. RNA prepared from hepatocyte monolayers, maintained for various lengths of time in culture, was translated in wheat germ extracts and Xenopus oocytes. Translation products were immunoprecipitated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In addition, total RNA from the cultured cells was hybridized to labeled cDNA probes to determine the number of sequences specific for each protein. Albumin mRNA was found to decrease with time in culture in all three assay systems, while transferrin mRNA remained constant. Analysis of the kinetics suggests a selective decay, with a half-life of 7 h, of the amount of albumin-specific RNA present at the beginning of culture. Apparently, in the hormone-deprived hepatocytes, there is little or no further transcription of the albumin gene. After addition of insulin to the cultures, albumin mRNA levels increased, suggesting an effect of this hormone on albumin gene utilization.

Hormonal regulation of fibrinogen synthesis in cultured hepatocytes.

Most of what was originally known of the effects of hormones on fibrinogen synthesis was based, as noted above, on experiments involving surgical removal of endocrine glands. Some caution should be exercised when using such in vivo experiments to derive the hormonal requirements of fibrinogen synthesis, however, since multiple hormonal alterations often occur in these animals. The development of a variety of ex vivo systems has allowed investigators to more carefully control the hepatocellular environment. The work of several laboratories, including our own, has now made it clear that hormones and other agents directly stimulate hepatocellular synthesis of fibrinogen. From the studies summarized here, using chick embryo hepatocytes as a model, several generalizations emerge: Fibrinogen synthesis may be considered to be a "constitutive" liver function, since hepatocytes cultured without serum, hormones or other macromolecular supplements synthesize this protein at a basal rate for several days. Addition of certain hormones (e.g. T3, dexamethasone, insulin), individually and in physiological concentrations, elicits an increase in fibrinogen production, varying with each agent in onset, dose, minimum exposure required and accompanying effects on the synthesis of other plasma proteins. Glucocorticoids and thyroid hormones are similar in the selectivity of their stimulation (neither affects albumin or transferrin synthesis) but differ in that thyroid hormones need to be present for just a short "triggering" period. The stimulation of fibrinogen synthesis by insulin occurs only following prolonged exposure to concentrations 10-times higher than the very low doses to which albumin synthesis responds rapidly.

Structural analysis of fibrinogen synthesized by cultured chicken hepatocytes in the presence or absence of dexamethasone.

Hepatocyte monolayers, derived from chick embryos and cultured in chemically defined medium without hormones, synthesize and secrete fibrinogen that resembles chicken plasma fibrinogen immunochemically and structurally. Addition of a synthetic glucocorticoid, dexamethasone, to the cultured cells resulted in an appreciable and relatively selective increase in fibrinogen synthesis. Autoradiography of fibrinogen that had been metabolically labelled with [35S]methionine and then subjected to SDS-polyacrylamide gel electrophoresis, unreduced or under disulfide-reducing conditions, revealed that only dimeric forms of fibrinogen, containing undegraded A alpha, B beta, and gamma chains, were secreted under stimulated and unstimulated culture conditions.

Serum stimulation of plasma protein synthesis in culture is selective and rapidly reversible.

Primary hepatocyte monolayers, derived from chick embryos, can be cultured from the onset in a completely chemically defined medium, free of added hormones. The liver cells synthesize and secrete a wide spectrum of plasma proteins for several days in this serum-free environment. Addition of fetal bovine serum elicits a 3-5-fold increase in the production of certain plasma proteins: fibrinogen, albumin, and the alpha1-globulin M. This effect of serum is selective; transferrin and plasminogen syntheses are enhanced less than 1.5-fold. Significant stimulation is observed with 0.1% fetal bovine serum, and half-maximal values for individual plasma proteins are obtained with concentrations ranging between 0.4 and 1%. The stimulatory activity of serum shows no developmental or species specificity. Plasma is active as serum derived from the same blood sample. The hepatocytes respond rapidly to serum, significant changes in albumin synthesis occurring less than 1 h after serum addition or removal. The effect of short exposure is fully reversible. These results establish the capacity of low concentrations of serum to stimulate plasma protein synthesis and underscore the importance of studying the effects of hormones and other factors under serum-free conditions. The findings suggest that, in addition to the classical hormones, ubiquitous but as yet uncharacterized serum components play a role in controlling this major hepatic function.